BackgroundTriple-negative breast cancer (TNBC) presents significant therapeutic challenges due to its immunosuppressive tumor microenvironment (TME). Oncolytic herpes simplex virus type 1 (oHSV1) offers dual mechanisms of tumor lysis and immune activation, yet the optimal cytokine payloads for TNBC remain undefined.MethodsWe developed a CRISPR/Cas9-mediated platform for high-efficiency oHSV1 engineering, replacing the ICP47 locus with murine IFN-gamma, GM-CSF, or IL-15R alpha/IL-15 fusion protein (IL15Fu). Constructs were validated for cytokine secretion, MHC modulation, and cytotoxicity in 4T1 TNBC and a panel of human cancer cell lines. Antitumor efficacy and immune remodeling were evaluated in a syngeneic 4T1 model using RNA sequencing and flow cytometry.ResultsThe CRISPR platform achieved 62.5-71.4% homologous recombination efficiency, enabling rapid virus construction. In vitro, OV-IFNG exhibited upregulated MHC I/II expression and potent cytotoxicity, while OV-GMCSF attenuated oncolysis in subsets of breast cancer cell lines. In the 4T1 model, OV-IL15Fu modestly improved tumor control and extended survival without apparent toxicity, while OV-IFNG induced early mortality associated with systemic toxicity. Transcriptomic profiling revealed divergent immune modulation: OV-IL15Fu enriched T cell/NK cytotoxicity pathways, OV-IFNG amplified cytokine/chemokine signaling, and OV-GMCSF paradoxically enhanced myeloid recruitment while inhibiting MHC-II pathways. Flow cytometry confirmed functional differences in immune activation: OV-IL15Fu expanding cytotoxic lymphocytes (CD8(+) T/NK cells), OV-IFNG preferentially promote Th1 polarization and innate immune activation, and OV-GMCSF failed to activate T cells despite myeloid infiltration.ConclusionsOur findings underscore the need for rational cytokine selection in oHSV1-based immunotherapy. While IFN-gamma increased immunogenic markers, its systemic toxicity and myeloid effects may limit benefit. GM-CSF exacerbated immune suppression in this context, whereas IL15Fu showed favorable immunostimulatory properties without detectable toxicity. These data support IL15Fu as a contextually promising payload for further evaluation in TNBC-targeted oncolytic virotherapy.
[1]Kunming Med Univ, Kunming, Yunnan, Peoples R China.
[2]Kunming Med Univ, Key Lab Affiliated Hosp 2, Kunming, Yunnan, Peoples R China.
(8.0+极速模式)